Does the nutrient stoichiometry of primary producers affectthe secondary consumer Pleurobrachia pileus?

Katherina L. Schoo Æ Nicole Aberle ÆArne M. Malzahn Æ Maarten Boersma

Received: 19 December 2008 / Accepted: 26 June 2009 / Published online: 8 July 2009

� Springer Science+Business Media B.V. 2009

Abstract We investigated whether phosphorus lim-

itations of primary producers propagate upwards

through the food web, not only to the primary

consumer level but also onto the secondary consum-

ers’ level. A tri-trophic food chain was used to assess

the effects of phosphorus-limited phytoplankton (the

cryptophyte Rhodomonas salina) on herbivorous

zooplankters (the copepod Acartia tonsa) and finally

on zooplanktivores (the ctenophore Pleurobrachia

pileus). The algae were cultured in phosphorus-

replete and phosphorus-limited media before being

fed to two groups of copepods. The copepods in turn

were fed to the top predator, P. pileus, in a mixture

resulting in a phosphorus-gradient, ranging from

copepods having received only phosphorus-replete

algae to copepods reared solely on phosphorus-

limited algae. The C:P ratio of the algae varied

significantly between the two treatments, resulting in

higher C:P ratios for those copepods feeding on

phosphorus-limited algae, albeit with a significance

of 0.07. The differences in the feeding environment

of the copepods could be followed to Pleurobrachia

pileus. Contrary to our expectations, we found that

phosphorus-limited copepods represented a higher

quality food source for P. pileus, as shown by the

better condition (expressed as nucleic acid content) of

the ctenophore. This could possibly be explained by

the rather high C:P ratios of ctenophores, their

resulting low phosphorus demand and relative insen-

sitivity to P deficiency. This might potentially be an

additional explanation for the observed increasing

abundances of gelatinous zooplankton in our increas-

ingly phosphorus-limited coastal seas.

Keywords Phosphorus limitation �Ctenophores � Ecological stoichiometry �Marine food webs � Gelatinous zooplankton �Trophic transfer

Introduction

Consumers habitually face the problem of nutritional

imbalances, as the nutrient content of their prey does

not always meet their requirements, leading to a

mismatch between supply and demand (Sterner and

Elser 2002). Specifically, in the relationship between

primary producers and herbivores, nutrients such as

K. L. Schoo (&) � N. Aberle � A. M. Malzahn �M. Boersma

Alfred-Wegener-Institut fur Polar- und Meeresforschung,

Biologische Anstalt Helgoland, Postbox 180, 27483

Helgoland, Germany

e-mail: Katherina.Schoo@awi.de

K. L. Schoo

Leibniz Institute of Marine Sciences, IFM-Geomar,

Dusternbrooker Weg 20, 24105 Kiel, Germany

M. Boersma

GKSS-Research Centre, Institute for Coastal Research,

Max-Planck-Straße 1, 21502 Geesthacht, Germany

123

Aquat Ecol (2010) 44:233–242

DOI 10.1007/s10452-009-9265-4

nitrogen (N) and phosphorus (P) are often limiting, as

there is a surplus of carbon in plants (White 1993).

Carbon is of course needed as an energy source, and

hence it will be respired through the food web, but as a

result many consumers will have more problems

meeting their nutrient requirements than their energy

requirements. Furthermore, the stoichiometry of

primary producers (i.e., the balance between different

nutrients) is highly variable due to the changing

balance between light and nutrients and often reflects

the changing nutrient availability of the environment

(Sterner et al. 1998). Therefore, for many consumers

food may be of varying but generally poor quality. In

contrast to their prey, animals are much more homeo-

static (i.e., they maintain a constant body composi-

tion), and their nutrient stoichiometry should therefore

be only weakly affected, if at all, by changes in the

nutrient stoichiometry of their food (Elser et al. 2000).

This herbivore homeostasis has led to the prediction

that food quality effects are not passed on to higher

trophic levels beyond the herbivore, as the nutrient

limitation of the primary producers would be buffered

by the herbivorous consumer (Sterner and Elser 2002).

Secondary consumers should subsequently not be

strongly affected by food quality at the primary

producer–herbivore interface. However, keeping

homeostasis is not without costs, and many studies

have shown that herbivores actually perform much

worse in food that is limited in phosphorus (Urabe

et al. 1997; Elser et al. 2001) or nitrogen (Van

Nieuwerburgh et al. 2004). Thus, the effect of nutrient

limitation results in lower densities and potential food

shortage for secondary consumers. Hence, the expec-

tation is that the low food quality experienced by

primary consumers should translate in a quantity effect

on higher trophic levels (Sterner and Elser 2002).

It is, however, becoming more and more apparent

that nutrient homeostasis of primary consumers is

less than perfect. Herbivorous zooplankters are not

always capable of maintaining a strict homeostasis

when faced with changes in nutrient stoichiometry of

their food source, and their nutrient content changes

accordingly (Malzahn et al. 2007a). Food quality

effects at the bottom of the food chain can thus

change the stoichiometry of the consumers and hence

may be passed on through the food chain as quality

effects. As an example, carbon to phosphorus ratios

of the freshwater cladoceran Daphnia can vary by a

factor of two (Plath and Boersma 2001; Boersma and

Kreutzer 2002). This implies that not only the

quantity but also the quality of zooplankters as food

source for their predators could be affected. Conse-

quently, higher trophic levels could in turn be faced

with food of different nutrient ratios, and the quality

effect of the primary producers could be transferred

higher up the food chain. This aspect has long been

neglected in the literature, and only very few studies

have addressed the possible effects of varying

phosphorus levels in primary producers on higher

trophic levels (Malzahn et al. 2007a; Boersma et al.

2008; Dickmann et al. 2008; Frost et al. 2008). Only

recently, Malzahn et al. (2007a) showed in an

experiment extending over three trophic levels that

phosphorus limitation on primary producers affects

the physiological condition of planktivorous fish

larvae and that severe phosphorus limitations in

algae can be traced to secondary consumers.

We therefore set out to further our knowledge on

nutrient limitations propagating upwards through the

food web in the form of food quality and investigate

the effects of phosphorus limitation in primary

producers on higher trophic levels. Hence, we studied

an artificial tri-trophic food chain, with the ctenophore

Pleurobrachia pileus as the secondary consumer. In

contrast to fish (from the aquaculture literature), not

much is known about the body composition of

ctenophores. Even though the work of Kremer and

co-workers is substantial when it comes to carbon,

nitrogen and biomolecules (Kremer 1977, 1982;

Kremer et al. 1986; Youngbluth et al. 1988), the only

study that we are aware of also including phosphorus

(Borodkin and Korzhikova 1991) suggest that the C:P

ratio of ctenophores can be as low as 83 (molar), and

hence the phosphorus requirement of P. pileus should

be high. Based on this, we expected that P-limited

copepods would be a food source of very poor quality

for Pleurobrachia pileus.

Materials and methods

We designed a tri-trophic experiment consisting of a

primary producer, the cryptophyte Rhodomonas

salina, a primary consumer, the copepod Acartia

tonsa, and a secondary consumer, the ctenophore

Pleurobrachia pileus. We chose P as the nutrient

under investigation as gradients of P-limitation are

stronger in algae growing in different media, and

234 Aquat Ecol (2010) 44:233–242

123

therefore experimentally easier to manipulate. More

importantly, however, is that P can be the limiting

nutrient in coastal seas (Elser et al. 2007; Vermaat

et al. 2008), and it has been observed that the

phytoplankton in the German Bight is P-limited in

summer (van der Zee and Chou 2005).

Primary producers

Stock cultures of the cryptophyte Rhodomonas salina

were cultivated in f/2 medium (Guillard and Ryther

1962). The water used during the experiment was

taken from the North Sea on one single occasion. The

water was filtered through a sterile 0.2 lm filter and

stored dark and cold until use. For the experimental

treatments, R. salina was cultured in nutrient replete

medium and in medium without added P. The

nutrient replete medium consisted of natural seawater

enriched with f/2 nutrients containing 36.3 lmol L-1

NaH2PO4, following Guillard and Ryther (1962). The

P-limited treatment consisted of f/2-enriched seawa-

ter without the addition of any phosphorus (–P). The

algae had access only to the P contained in the natural

seawater at the time of filtration (1.4 lmol L-1).

Experimental algae were kept at 17�C under a 16:8 h

light:dark (L:D) cycle.

Preliminary tests on algal growth rates under

experimental conditions showed P limitation of R.

salina with the P-deficient medium after 4 days. Algal

densities in the stock solution were determined with a

CASY cell counter (Scharfe System CASY Cell

Counter and Analyser System). To ensure constant

food quality, new cultures of R. salina were inoculated

daily for both treatments with a starting concentration

of 0.2 9 106 cells mL-1 for the f/2 treatment and

0.3 9 106 cells mL-1 for the –P treatment. After the

predefined growth period of 4 days, algae were

harvested at densities of *1.5 9 106 cells mL-1 for

the f/2 treatment and 1.0 9 106 cells mL-1 for the –P

treatment.

Primary consumers

Copepod eggs were obtained from a laboratory culture

of the calanoid copepod Acartia tonsa. For the

production of eggs, animals were kept in filtered

natural seawater (salinity 31) in a 200-L cylindrical

tank on a 16:8 h L:D cycle at 18�C. The copepods were

fed a mixture of the algae Rhodomonas salina and the

heterotrophic flagellate Oxyrrhis sp. Eggs were

siphoned off the bottom of the tank daily and stored

in an airtight container in the dark at 4�C until use.

When needed, these eggs were incubated in fresh

seawater in 4-L plastic bags at a density of about

3,000 individuals per litre. The hatching rate was

around 25%. Copepods were first fed 24 h after

hatching, 48 h after the addition of the eggs to the

water in the plastic bags. In order to avoid changes in

the phosphorus content of the algae during their

incubation with the copepods, the eggs were incu-

bated in phosphorus-free artificial seawater, adjusted

to a salinity of 31 (salt: hw Marinemix, www.hw-

wiegandt.de). Copepods were fed 10,000 cells of

Rhodomonas per individual and day, which is con-

sidered to be ad libitum for larval stages

([1 mg C L-1). Copepods were fed the same amount

of algal cells for each treatment to avoid food quan-

tity effects. To guarantee a steady supply of food at

constant quality for the secondary consumer, the

ctenophore Pleurobrachia pileus, two new bags of

copepods were started each day.

The copepods grown on P-limited algae displayed

a delayed development resulting in a time-lag of

*1 day when compared to the copepods reared on

f/2 algae. Therefore, P-limited copepods were har-

vested on day 8 after hatching, when the majority had

reached the sixth naupliar stage, and f/2 copepods

were 1 day younger. This ensured that copepods from

different treatments were in the same developmental

stage and had the same size. Copepods were fed to

the ctenophore along a P gradient at a concentration

of 0.5 individuals mL-1. This amount is above the

densities reported for food saturation in ctenophores

(Greve 1972; Reeve et al. 1978; Gibbons and

Painting 1992), which was confirmed by the fact that

some copepods were still present in all ctenophore

containers 24 h after feeding.

Secondary consumer

The ctenophore Pleurobrachia pileus was obtained

from Helgoland Roads, North Sea (54�110 N, 7�530 E).

P. pileus is an ambush predator, catching its motile

prey in lateral filaments on its tentacles (Greve 1970;

Gibbons and Painting 1992). This feeding mechanism

is unselective for actively swimming prey (Fraser

1970). Individuals were transferred to flow-through

tanks and kept at ambient water temperature in filtered

Aquat Ecol (2010) 44:233–242 235

123

seawater. Individuals were starved for 5 days prior to

the first feeding in the experiment. Only ctenophores

of the same size range (10–15 mm) were used in the

experiment. For the duration of the experiment, each

individual P. pileus was kept in a separate 1 L glass

bottles containing filtered seawater. Water was

changed daily prior to feeding to remove most of

the uneaten food organisms. P. pileus were fed

copepods along a P-gradient, resulting in five treat-

ments. The treatments were randomly assigned to 50

ctenophores, ten replicates for each of the five feeding

treatments. The copepods were mixed prior to being

fed to P. pileus, resulting in the following regimes: at

the extreme ends of the P-gradient the ctenophores

were fed only copepods from the f/2 or the –P

treatment, respectively; three other treatment groups

received 75, 50 and 25% of the f/2-reared copepods,

and 25, 50 and 75% of –P-reared copepods to result in

the amount of 500 copepods per ctenophore day-1.

P. pileus were fed daily for 9 days, after which the

ctenophores were harvested and first frozen at -80�C

and thereafter freeze-dried, weighed and kept in a

desiccator until analysis.

Analytical procedures

For the analysis of carbon content of the algae,

*4 9 106 cells were filtered onto pre-combusted and

washed Whatman GF/F filters. For the analysis of

copepod carbon, 50 individuals were counted into tin

capsules. The carbon content of the samples was

measured with a Fisons EA 1108 CHN analyser.

Phosphorus was analysed as orthophosphate, accord-

ing to the method described by Grasshoff et al. (1999),

following oxidative hydrolysis. The samples were

treated with an oxidation agent (K2S2O8, H3BO3,

NaOH in distilled water) under high pressure and at

high temperature (120�C) in an autoclave to convert

the phosphorus compounds to the ortho-phosphate

form. Molybdate-antimony-solution (containing ammo-

nium molybdate (NH4)6Mo7O24 9 4H2O, antimony

potassium tartrate K(SbO)C4H4O6 9 0, 5H2O) and

ascorbic acid were added, and the P-content mea-

sured photometrically. For the analysis of carbon and

P-content of Pleurobrachia pileus, pulverized tissue

homogenate was used.

Dry weight (or somatic growth) is not a useful

response variable in this case for several reasons.

First, as we could not work with a cohort of animals

of completely equal size and age, initial weight could

not be established accurately enough, and hence the

variation in computed growth rates would probably

be much larger than any expected effect. Secondly,

dry weight in gelatinous zooplankton is mainly

determined by the salt in the water of the body

cavities, thus does not give an accurate estimate of

the growth in biomass. Consequently, we used the

carbon content per dry weight as an indicator for

physiological condition. Animals with a higher C to

dry weight ratio have a better physiological condi-

tion, as was observed by for example Daly (2004).

Furthermore, the nucleic acid content of the animals

was also established. The techniques to determine the

amounts of RNA and DNA in animal tissue are well

established, and using the amount of nucleic acids

RNA and DNA in the individual organisms as a

measure of overall physiological condition, and as a

proxy for growth, is commonly used in fisheries

biology (e.g., Clemmesen et al. 2003). In many

studies, a ratio between the RNA and the DNA

content of animal tissue is used, under the rationale

that DNA content as a constitutive component of the

cell should be more constant relative to RNA, which

should be higher under active growth. A high RNA to

DNA ratio is therefore indicative of growth and a

good physiological condition. Ctenophores are

known to react to adverse conditions by changes in

size (Kremer 1977). This means that most likely, they

lose complete cells, with both DNA as well as RNA

content. Indeed, recent work by Hamer (2008)

showed that the RNA:DNA ratio in the ctenophore

Mnemiopsis leidyi was more or less constant, even

after many days of starvation, whereas the concen-

trations of RNA and DNA were much more respon-

sive. Therefore, in this study we used the absolute

amounts of RNA and DNA in the tissue of the

individuals as indicators for animal physiological

condition (Ferron and Leggett 1994; Parslow-Wil-

liams et al. 2001; Gorokhova 2003). The method used

for the nucleic acid analysis was from Clemmesen

et al. (2003) with some modification after the

determination of the protocol for the extraction of

nucleic acids from ctenophore tissue.

Freeze-dried tissue was pulverized using a

pellet pestle and rehydrated in 400 lL Tris–SDS

buffer (Tris 0.05 mol L-1, NaCl 0.01 mol L-1,

EDTA 0.01 mol L-1, sodium dodecyl sulphate

(SDS) 0.005%) for 25 min. Glass beads (2 mm and

236 Aquat Ecol (2010) 44:233–242

123

0.17–0.34 mm diameter) were added, and the tubes

shaken in a Retsch MM 301 cell-mill for 15 min. The

homogenate was centrifuged (Sartorius Sigma

3–18 K; 8 min, 3,800 g, 4�C), and 130 lL of the

supernatant used for analysis.

The amount of nucleic acids was determined

fluorometrically in a microtiter fluorescence reader

(Fluoroskan Ascent) using the fluorophor ethidiumbr-

omide (EB). Total nucleic acids were measured first.

Subsequently, RNAse was added to the samples in

order to digest the RNA. After the enzyme treatment

(30 min at 37�C), the remaining DNA was measured.

The RNA fluorescence was calculated by subtracting

the DNA fluorescence from the total nucleic acid

fluorescence. RNA calibrations were set up at each

measurement day. The DNA concentrations were

calculated using the relationship between RNA and

DNA fluorescence described by Le Pecq and Paoletti

(1966).

Results

Primary producer and primary consumer

Algal molar C:P ratios varied significantly between

treatments (f/2 *230, –P *430; ANOVA F1,18 =

10.75; P \ 0.004; Fig. 1). The results confirm that

the –P growth medium really was limited in phos-

phorus; the natural seawater contained just enough P

to enable the algae to grow. The molar C:P ratio of

the copepods also showed differences between

treatments, which were significant at P = 0.07

(f/2 *140, –P *195; ANOVA F1,10 = 4.06;

P = 0.07; Fig. 1).

Secondary consumer

The differences in the C:P ratios of P. pileus cultured

on the extremes of the food gradient were not

significant (Fig. 1), but were significantly higher

than those for their food (ANOVA F1,17 = 6.60;

P = 0.02), which was mainly caused by the differ-

ences between f/2 copepods and ctenophores, with

much lower C:P values for the copepods. As expected

for the reasons mentioned earlier, dry weight of

Pleurobrachia pileus was not significantly affected

by the food they received (Fig. 2a), nor was there a

clear pattern of C:P ratios of the P. pileus individuals

over the gradient (Fig. 2c).

P. pileus fed a diet consisting solely of P-limited

copepods (0% treatment) showed the highest values

of carbon per dry weight (Fig. 2b; linear regression,

y = 4.253-0.015x; r2 = 0.36; P = 0.001). Further-

more, both the DNA content (lg mg DW-1) (Fig. 2d;

linear regression, y = 0.294-0.002x; r2 = 0.18;

P = 0.01) and the RNA content of the experimental

animals (Fig. 2e; linear regression, y = 1.249-

0.009x; r2 = 0.18; P = 0.01) showed a significantly

negative relationship with the percentage of f/2

copepods in their food. The content of RNA and

DNA in individuals were highly correlated

(r2 = 0.98). These results indicate a simultaneous

loss of both nucleic acids related to the amount of f/2

copepods in their diet (Fig. 2 d, e), and hence the

ratio between RNA and DNA concentration of the

experimental P. pileus individuals did not show

significant change over the experimental gradient

(Fig. 2f). The RNA content (lg mg DW-1) of the

animals in the starvation group (mean value 0.4 lg

RNA mg DW-1) was lower than that of the fed

individuals. No significant correlation was found

between dry weight of the animals and their bio-

chemical composition (data not shown).

Even though the amount of explained variation in

the significant relationships is modest, all physiolog-

ical condition indicators point in the same direction:

copepods that were grown on P-limited algae repre-

sent a food source of higher quality for Pleurobrachia

pileus compared to those fed P-replete algae.

0

100

200

300

400

500

600

700

f/2

-P

C:P

rat

io (

mol

ar)

R. salina A.tonsa P. pileus

*

*

Fig. 1 Molar C:P ratios of Rhodomonas salina (n = 10),

Acartia tonsa (n = 7) and Pleurobrachia pileus (n = 7) in a

nutrient-replete (f/2) and phosphorus-limited (–P) environment.

Asterisk marks significant difference (P \ 0.05 for R. salina,

P = 0.07 for A. tonsa) from the other treatment of the given

species. Error bars: standard deviation

Aquat Ecol (2010) 44:233–242 237

123

Discussion

The classical picture in the current literature is that

when going up in the food chain the C:P ratio of the

organisms in each level is lower than that of the level

below, as carbon is lost through respiration and

excretion (Sterner et al. 1998). Here, we present an

unusual case where the C:P ratio of the predator

(P. pileus) actually is higher than that of the prey. As a

consequence, the f/2 copepods in this study may have

been a lower quality food source for the predator

P. pileus, not because they contained too much carbon

Dry

wei

ght (

mg

ind-1

)

0

2

4

6

8

10

12

14C

:P r

atio

50

100

150

200

250

300

350

400

450

Percentage f/2 copepods in food for Pleurobrachia pileus0 20 40 60 80 100 0 20 40 60 80 100

0 20 40 60 80 100 0 20 40 60 80 100

0 20 40 60 80 100 0 20 40 60 80 100

RN

A c

onte

nt (

µg m

g D

W-1

)

0

1

2

3

4

C c

onte

nt (

mol

mg

dry

wei

ght-1

)

0

1

2

3

4

5

6

Rat

io2

3

4

5

6

7

DN

A c

onte

nt (

µg m

gDW

-1)

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

Y= 4.253 - 0.015X, (r2 = 0.36; p=0.001)

Y= 1.249 - 0.009X, (r2 = 0.18; p = 0.01)

Y= 0.294 - 0.002X, (r2 = 0.18; p = 0.01)

(a) (b)

(c) (d)

(e) (f)

Fig. 2 Reactions of P. pileus to food of different P content.

Regression lines indicate significant relationships between the

percentage of f/2-fed copepods in the diet of P. pileus and

the response variables. a Dry weight of P. pileus after the

incubation period; b carbon content (mol per mg dry weight); cC:P ratio (molar) of the experimental P. pileus; d DNA

content; e RNA content; and f the ratio between RNA and

DNA content in P. pileus

238 Aquat Ecol (2010) 44:233–242

123

relative to phosphorus, but rather the reverse, and it

does explain why we find the best physiological

condition of the P. pileus individuals on copepods-fed

P-limited R. salina. Most likely, given the high C:P

values of P. pileus, even the low-P copepods contained

too much phosphorus. This excess in P resulted in a

diet poor in carbon, making the ctenophores poten-

tially energy limited, rather than P limited (see also

Gaedke et al. 2002). So, the hypothesis based on the

study of Borodkin and Korzhikova (1991), that due to

their relatively high phosphorus content Pleurobra-

chia, or perhaps even all ctenophores, is expected to

show phosphorus limitation quickly, clearly needs to

be rejected. In fact, several studies (Schneider 1989;

Anninsky et al. 2005) have indicated a very high

content of proteins in ctenophores, which is corrobo-

rated by the low C:N values found by Kremer and co-

workers (Kremer 1977, 1982; Kremer et al. 1986;

Youngbluth et al. 1988), so the measurements of

Borodkin and Korzhikova (1991) for Mnemiopsis

leidyi might be considered unusual and not reflect a

general pattern in ctenophores.

Virtually no information exists on the nutrient

stoichiometry and physiology of ctenophores, and of

P. pileus in particular. Hence, we can only speculate

on the physiological processes that underlie the

patterns that we have observed. Ctenophores are

highly opportunistic predators that feed when possible

but can withstand long periods of starvation, partic-

ularly in temperate regions (Greve 1972), and respi-

ration rates go to virtually zero under starvation

(Hamer, personal communication). The fact that these

mechanisms are in place suggests that carbon limita-

tion is not uncommon in ctenophores. Furthermore,

the only study that we are aware of reporting on

excretion rates of carbon and phosphorus in cteno-

phores (Kremer 1977) reported very high turnover

rates of Mnemiopsis leidyi for phosphorus (20–50%

day-1)¸ also suggesting that phosphorus is not the

limiting nutrient in ctenophores. Based on this, we

would not really expect to see an effect of the changed

nutrient stoichiometry, as obviously even P-limited

copepods contain sufficient phosphorus to sustain an

optimal physiological condition in P. pileus. We do,

however, see a decrease in condition of the cteno-

phores with increasing phosphorus content of their

prey. Boersma and Elser (2006) hypothesized that any

excretion of surplus nutrients comes with a cost.

Traditionally, most studies on zooplankton feeding

(Sterner and Hessen 1994; Urabe et al. 1997; DeMott

et al. 1998; Sterner and George 2000; Darchambeau

et al. 2003) have focused on the effect of high C:P

food on growth and reproduction and explained lower

performance on high C:P food with the need for the

animal to rid itself of excess carbon to meet the

P demand. When we follow Boersma and Elser (2006)

and accept that also excreting excess phosphorus

comes with a cost, we can explain the patterns found

in this study. Based on our results, we suggest that the

phosphorus content of the f/2 copepods may have

been too high and that the excretion of the excess

phosphorus could come at the cost of a reduced

physiological condition.

The analysis of the RNA and DNA contents in

animals has been utilized as an indicator of the fitness

and physiological condition of animals (Clemmesen

et al. 2003; Melzner et al. 2005; Malzahn et al.

2007b). The method for the analysis of the RNA and

DNA concentrations used here was adapted from

Clemmesen et al. (2003) after the best conditions for

nucleic acid extraction from ctenophore tissue were

determined. Both the amount of tissue used and to a

more important extent the concentration of the

detergent in the buffer influenced the results. Extrac-

tion of nucleic acids from the tissue of P. pileus

proved difficult, as the yield was very low and

unstable at first. By testing a range of combinations of

different sample weights and buffer concentrations, a

procedure enabling a stable and consistent extraction

of nucleic acids was established. The most stable and

reliable results were obtained with a combination of

high tissue sample weights and a reduced concentra-

tion of the buffer SDS, from the 0.01% used in the

protocol established by Clemmesen et al. (2003) to

0.005%. Our results show that RNA and DNA

content were highly correlated (r2 = 0.98). Both the

DNA content as well as the RNA content was

affected in parallel as a result of the different feeding

environment. Ctenophores react to adverse feeding

situations by decreases in body size (Kremer 1977);

the underlying mechanism to this seems to be the

shedding of whole body cells (Hamer 2008), reducing

the total amount of cells and including the loss of

RNA and DNA contained in these cells. As a result,

the ratio of RNA to DNA content in ctenophore tissue

is not a very useful measure to assess physiological

condition, as was already expected from the fact that

body size is so plastic in ctenophores.

Aquat Ecol (2010) 44:233–242 239

123

Obviously, this experiment represented a very

artificial world, with organisms that might not even

meet in the field. However, our results have the

power to reveal mechanisms that would not be visible

in the field and certainly may be of relevance. Having

said this, ctenophores obviously do not feed exclu-

sively on juvenile copepods as was the case in this

study. Ontogenetic changes in the nutrient stoichi-

ometry and the phosphorus content in particular have

been reported by Carrillo et al. (2001) and Villar-

Argaiz et al. (2002). The P content of copepods (the

calanoid copepod Mixodiaptomus laciniatus) was

observed to have changed over the course of the life

cycle, with nauplii having a higher P content than

adult copepods. So the results might have been

different if we had used adult copepods as prey for

P. pileus. Furthermore, 1-L bottles are certainly fairly

small for P. pileus, and the organisms might have had

problems developing their tentacles. However, as all

experiments were carried out in the same sized

vessels, this should not have been a problem.

Potential implications

By feeding the copepods to the ctenophores along a

gradient, we were able to show that more subtle

phosphorus limitations can also have an effect on

consumers and are still traceable to the next trophic

level. This is of particular importance during transi-

tional phases in the phosphorus content of seawater,

such as the late phase of a phytoplankton spring

bloom in temperate regions. During this time, phy-

toplankton becomes more and more phosphorus-

limited, and as a result the zooplankters feeding on

them will also most likely change their nutrient

composition to some extent. In this special case, P-

limited copepods represent a food source of higher

quality for P. pileus. Interestingly, but with a great

need for further study, this period of phosphorus

depletion in phytoplankton is exactly the time when

we see high densities of Pleurobrachia pileus around

Helgoland (Greve et al. 2004). It is possible that the

ctenophores, being less vulnerable to the P-limited

situation, are at an advantage during this time.

Currently, many coastal seas are undergoing re-

oligotrophication as a result of decreasing phosphorus

inputs from the rivers (Wiltshire et al. 2008). This

change in the nutrient composition towards a more

phosphorus-limited regime may result in food of high

quality for the ctenophores. In turn, Malzahn et al.

(2007a) showed that P-deficient copepods are inferior

food for zooplanktivorous larval fish, which utilize the

same food source. One might speculate that this

mechanism could further support the general trend

towards a more gelatinous North Sea as reported by

Attrill et al. (2007) as a phosphorus-limited environ-

ment would favour the occurrence of ctenophores in

the plankton. Furthermore, the effects of nutritional

imbalances may even be increased by rising CO2

levels, which are predicted by future climate change

scenarios (IPCC 2007). This could possibly lead to

changing stoichiometric composition of primary pro-

ducers, which has consequences for consumers per-

formance (Urabe et al. 2003) and might lead to higher

C:P ratios of primary producers. It has been forecast

that in phosphorus-deficient systems zooplankton

with high C:P ratios, such as P. pileus, will become

more important (Gaedke et al. 2002). From this, one

would predict that ctenophores will continue to grow

in their importance in many coastal ecosystems.

The results presented here show that phosphorus

limitations in primary producers can be traced to

primary and secondary consumers. The ensuing

quality effects propagate through the food web and

affect higher trophic levels, albeit in a different

manner than originally expected. The effects of the P

limitation, as such, on P. pileus were rather weak, but

the changed nutrient composition of the algae

affected the secondary consumer nevertheless. Con-

trary to our original expectations, our results show

that the ctenophore Pleurobrachia pileus is not

negatively affected by low P food and may even

benefit from a diet of copepods feeding on P-deficient

phytoplankton.

Acknowledgments This study is funded by the German

Science Foundation (DFG AB 289/2-1) and is part of the AWI

Food Web project. We thank all colleagues in the Food Web

projects and three anonymous reviewers for stimulating

discussions and helpful comments to improve earlier versions

of this manuscript.

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